PROJECT SUMMARY G protein-coupled receptors (GPCRs) are well known for their ability to convert extracellular information encoded in hormones, odorants, and peptides into rapid changes in cellular homeostasis by modulating the function of effector enzymes and channels in the cell. However, analysis of human cancer genomes indicates that some GPCR signaling pathways lead to sustained signals that in pathological settings are linked to tumor growth and metastasis. A common feature of such pathways is the activation of Rho GTPases by Dbl family Rho guanine nucleotide exchange factors (RhoGEFs). The TrioC subfamily (composed of Trio, Kalirin, and p63RhoGEF) is directly activated by heterotrimeric G?q subunits and is strongly implicated in ocular melanoma and leukemia. The P-Rex subfamily (composed of P-Rex1 and 2) is activated synergistically by direct interactions with G?? and the lipid PIP3, and is overexpressed in many breast and prostate tumors where it plays a metastatic role. Trio and P-Rex1/2 have thus emerged as important chemotherapeutic targets. The fact that the molecular and cellular mechanisms underlying regulation of these enzymes are as of yet poorly understood prevents a rational approach to the design of novel therapeutic approaches. By determining crystallographic and cryo-EM structures of members from these RhoGEF subfamilies, this proposal seeks to define molecular mechanisms of activation and to test these hypotheses through a battery of functional assays. In parallel, we extend our functional analysis into model cell systems to understand how constitutively active heterotrimeric G proteins and GPCRs promote cancer cell growth (as in ocular melanoma) and metastasis (as in breast cancer) via TrioC and P-Rex subfamily RhoGEFs. By understanding the basis for regulation of RhoGEF activity by heterotrimeric G proteins both in vitro and in relevant cellular contexts, we will accelerate discovery of new biological insights and novel therapeutic strategies that can be used to combat cancer.